The purpose of this project is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. During the coming grant period the major emphasis will be on the use of 13C, 15N, and 18O isotope effects: 1) Isotope effects in the beta-lambda bridge and lambda-nonbridge oxygens of ATP will be used to study the reactions catalyzed by creatine and arginine kinases. Secondary 18O isotope effects on the hydrolysis of a phosphotriester containing a cyclic ethylene group and a leaving group with pK 8.6 will tell whether the phosphorane intermediate is neutral or monoanionic. 2) The mechanism of asparagine synthetases A and B from E. coli will be studied with 13C, 15N and 18O isotope effects to determine the rate limiting steps in the chemical mechanism. 3) The mechanism of isocitrate dehydrogenase will be studied with 13C and deuterium isotope effects to determine the rate limiting steps with alternate substrates and mutant enzymes. 4) 13C and 18O isotope effects will be used to determine the relative rates of the steps in the reactions catalyzed by dTDP-glucose-and CDP-glucose 4,6-dehydratases. 5) 13C, 15N and 18O isotope effects will be used to determine the mechanism and relative rates of the steps in the urease-catalyzed hydrolysis of formamide. 6) The compounds giving the upfield 31P NMR signals that are thought to represent mono-, di- or tricarboxy-P in solutions of phosphate in DMF or DMSO containing CO2 will be characterized and tested as substrates for appropriate enzymes. 7) 13C and 15N isotope effects will be measured on the reaction catalyzed by glycosylasparaginase, using a series of substrates containing 4'-substituted phenyl groups on the beta-nitrogen of asparagines.